Gene regulatory processes are fundamental in most, if not all, forms of disease, as well as in all of developmental biology. Accordingly, a primary goal of modern medicine is to understand gene regulation and to identify specific modulators of gene expression. These modulators serve as antineoplastic agents, antiviral agents, antifungal agents, and the like, for the treatment of a very wide variety of diseases.
Assays for monitoring gene expression are well known, including northern blotting, RT-PCR, RNase and S1 protection, reporter gene expression (e.g., chloramphenicol transferase (CAT) assays), etc. General texts that describe assays for monitoring gene expression include Sambrook et al., Molecular Cloning--A Laboratory Manual (2nd ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1997) ("Ausubel"). Specialized apparatuses for performing these assays are commercially available.
In addition to these standard methods typically used to monitor gene expression, specialized formats for detection of nucleic acids are also available. For example, Coutlee et al. (1989) Analytical Biochemistry 181:153-162 describe non-isotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody which binds DNA:RNA hybrids. In these assays, hybridization of an RNA target with a biotinylated DNA probe is performed, followed by incubation of the hybridized target-probe duplex on an anti-biotin plate, reaction of the resulting bound duplex with a .beta.-galactosidase labeled monoclonal antibody specific for RNA-DNA hybrids, and addition of a fluorescent substrate. In another example, a "sandwich" hybridization method is described for non-isotopic detection of RNA using oligonucleotides (Ishii & Ghosh (1993) Bioconjugate Chem. 4:34-41). In these assays, the RNA target is hybridized to a first complementary oligonucleotide, which is linked to a bead. The RNA target is then hybridized to a second complementary oligonucleotide conjugated to alkaline phosphatase. The RNA target is detected by providing a chemiluminescent alkaline phosphatase substrate.
Other investigators have also reported immunological detection of DNA:RNA hybrids, including Bogulavski et al. (1986) J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407; Rudkin (1976) Nature 265:472-473, and Stollar (1970) PNAS 65:993-1000. Similarly, detection of DNA:DNA hybrids and RNA:RNA hybrids has also been described. See, Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650, and Stollar (1970) PNAS 65:993-1000. Stollar (1970) and Rudkin (1976) (both supra) showed that DNA:RNA hybrids in solution can be captured on plastic or nitrocellulose supports coated with an anti-poly(A)-poly(dT) polyclonal antibody. A monoclonal antibody against DNA:RNA heteroduplexes and RNA:RNA hybrids, which does not recognize DNA duplexes, single-stranded DNA or single-stranded RNA, has been prepared and characterized. See, e.g., Bogulavski et al. (1986) J. Immunol. Methods 89:123-130; Viscidi et al. (1988) J. Clin. Microbiol. 41:199-209, and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12. This antibody was used for the detection of DNA:RNA hybrids immobilized by binding of the DNA component of the duplex to a nylon bead, or to avidin latex. Immunoassays for detecting nucleic acids have been adapted to in vitro qualitative detection of human papillomavirus (HPV), e.g., for use in detecting cervical abnormalities. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).
One strategy for identifying pharmaceutical lead compounds is to develop an assay that provides appropriate conditions for monitoring the activity of a therapeutic target for a particular disease. This assay is then used to screen large numbers of potential modulators of the therapeutic target in the assay. For example, libraries of chemical compounds can be screened in solid phase assays using robotic components. Although the immunoassay-based and sandwich-based nucleic acid detection strategies described above have been useful for detecting DNAs, RNAs, and DNA:RNA hybrids, they have not been adapted to high throughput gene expression monitoring assays that could be used for screening for pharmaceutical lead compounds. Such high throughput assays for expression monitoring and pharmaceutical compound screening are desirable. This invention provides these assays, as well as other features which will become apparent upon review.